PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and affects up to 45,000 people annually within the United States, with a 5-year survival rate of 57%. Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, resulting in dramatic anti-tumor responses in some HNSCC patients; however, only about 20% show any survival benefit, and of these it is not clear what predicts response. ICIs have been thought to work primarily through reactivation of exhausted cytotoxic CD8+ T cells (CD8s) residing within the tumor microenvironment of solid tumors. Recent work, however, indicates that various CD4+ T cell populations outside the tumor microenvironment (peripheral immune system) likely play critical roles in carrying out anti-tumor immune responses. Given increasing evidence that immune cells other than CD8s are important for anti-tumor immunity, it is critical to identify and characterize these cells in order to understand their roles during HNSCC tumorigenesis and ICI therapy, which are currently unknown. This proposal by Dr. Kyle Jones, DDS, PhD seeks to significantly advance our understanding of cancer immunology in the context of HNSCC tumors, with the ultimate goal of improving existing ICI therapies and developing new treatment strategies. With the assistance of a multi-disciplinary mentoring team, he will use a novel systems biology approach to accomplish the following Aims: (AIM 1) Quantify the effects that HNSCC tumors have on systemic immunity and determine if these changes are conserved across tumor, blood, and regional lymph nodes; (AIM 2) Determine how the ICI atezolizumab alters the intratumoral and peripheral immune states of HNSCC patients and if these changes are associated with tumor response to therapy. These Aims will define how HNSCC tumors alter the composition and function of patients' immune systems at single cell and systems levels as well as identify/characterize the specific immune cells that contribute to anti-tumor immunity following ICI therapy. Dr. Jones will use high throughput single-cell methods such as mass cytometry, multiplexed ion beam imaging, and single-cell RNAseq along with established analytic pipelines on HNSCC patient specimens obtained before and after ICI therapy. The innovation of this approach, which permits simultaneous study of all immune cells, including CD4+ and CD8+ subsets, will reveal how changes in any individual immune cell population affects the overall immune state. It will also be important for the discovery of additional immune cell populations and biomarkers associated with HNSCC anti-tumor immunity following ICI therapy. Dr. Jones's long-term career goal is to become an expert, leader, and independent translational scientist in the fields of head and neck cancer immunology and immunotherapy. Through this K23 award, he will obtain the additional training in experimental immunology, single cell analysis methods, computer programming, advanced statistical methods, and career development he needs to successfully achieve this goal.